In order to enhance the economies of protein production in microorganisms, there have been substantial efforts to improve the efficiency of transcription and translation, maximize the proportion of total protein directed to production of the desired product, enhance the viability of the modified host, and improve the efficiency with which the modified host may be obtained. Using strong promoters to express heterologous genes in appropriate hosts is a major strategy in biotechnological applications. The glyceraldehyde-3-phosphate dehydrogenase (GPD, EC 1.2.1.12) promoter is a strong constitutive promoter which can be induced by any carbon source and has been widely used in the expression of heterologous proteins in Saccharomyces cerevisiae, Pichia pastoris and other yeasts.
GPD is one of the key enzymes in the glycolytic and gluconeogenesis pathway and comprises up to 5% of the soluble cellular protein content in S. cerevisiae and other higher eukaryotes. Furthermore, gpd mRNA accounts for 2˜5% of the poly (A)+ RNA present in yeasts. These observations suggest that the gpd gene is regulated by a highly active promoter. In fact, vectors carrying the homologous gpd promoter region have been reported to be efficient in directing expression of heterologous genes in yeasts (Bitter and Egan, 1988, Doring et al., 1998, Eriksson et al., 1995, Vassileva et al., 2001) and filamentous fungi (Juge et al., 1998, Punt et al., 1987). However, known expression vectors containing genetic regulatory elements for expression in filamentous fungi of the ascomycetes class cannot be efficiently expressed in filamentous fungi of the basidiomycetes class.
The gpd genes have also been cloned from basidiomycetous fungi, including Schizophyllum commune, Phanerochaete chrysosporium, Agaricus bisporus (Harmsen et al., 1992), and Lentinula edodes (Hirano et al., 1999). Among these mushrooms, genetic transformation using homologous gpd promoter was reported successful only in A. bisporus, Flammulina velutipes and L. edodes (Hirano et al., 2000, Kuo et al., 2004, van de Rhee et al., 1996). Although heterologous promoters have been used for the expression of drug-resistant marker genes, the genetic transformation is not sufficient to express heterologous genes (Ruiz-Diez, 2002). To sufficiently and effectively express a heterologous gene, it is important for a host cell to recognize the promoter sequence by its transcriptional machinery. Chun-Yi Kuo et al. demonstrated that a heterologous gene, hygromycin B phosphotransferase gene (hpt), can be expressed in F. velutipes (Kuo et al., 2004). However, it was found that the gpd genes in some basidiomycetous fungi, though highly similar, are significantly different in their promoter regions.
The Pleurotus is a well-known edible mushroom. The term “Oyster Mushroom” applies equally to Pleurotus ostreatus, Pleurotus pulmonarius (which is often paler and appears in the summer), and Pleurotus populinus (which is found on the Quaking Aspen). The Pleurotus occupies the third position in the worldwide market of industrially produced mushrooms. In addition, the Pleurotus produces various secondary metabolites of medical interest.
There is a need to increase the economical and industrial values of the Pleurotus by improving the expression of heterologous genes.